Bafilomycin-like metabolite from a novel micromonospora species

ABSTRACT

The invention provides a compound of formula (I) and pharmaceutically acceptable salts thereof. Processes for the isolating of this bafilomycin metabolite from a novel  Micromonospora  species, pharmaceutical compositions containing this metabolite and methods of treatment using said metabolite are also described

The present invention relates to a novel bafilomycin-like metaboliteisolated from a novel micromonospora species having potentantiproliferative activity as well as to processes for isolating saidmetabolite, pharmaceutical compositions containing this metabolite andmethods of treatment using said metabolite.

Although the increasing basic knowledge of major cellular signalingpathways has allowed recent discovery of promising “targetedtherapeutics” to help fighting cancer (e.g., inhibitors offarnesyltransferase, growth factor receptor tyrosine kinases, Bcr/abl,etc . . . ), more classical cytotoxics still remain an important part ofthe foundation of an oncology product portfolio in the modempharmaceutical industry. The partial success of traditional cancercytotoxic chemotherapy stems in large part from agents derived fromnatural sources.

Macrolides are a group of antibiotics that includes natural members.Most of the macrolides are produced by Streptomyces spp. bacteria.However also macrolides produced by Micromonospora spp. are known.Macrolides are characterised in that they are composed of a largeaglycone ring (from 14-16 carbon atoms) to which are attached severalsugars. A special series of these macrolides are the bafilomycins. Thesecompounds share the structural feature of having a 16-memberedmacrocyclic lactone ring and one or two side-chain sugars. Theaforementioned bafilomycins were previously known to have activity asantiparasitics, antithelmintics, antibiotics, insecticides, antifungals,agents for the therapy of bone diseases and as vacuolar-type ATPaseproton pump (v-ATPase) inhibitors. Later on, it has been found that,when run through whole tumour-cell assays, compounds from this groupexhibit antitumour activity.

WO 01/02413 discloses bafilomycin derivatives of formula (II),

wherein R is —H or —CO—CH═CH—COOH, and pharmaceutically acceptable saltsand esters thereof. The compounds display activity against tumour growthin mammals. WO 01/02413 further provides a method for the treatment orprophylaxis of tumour invasion by administration of compounds of formula(II) or of known macrolide compounds.

The present invention encompasses a novel structure of formula (I), atautomer thereof, or a pharmaceutically acceptable salt thereof, with anew hexadiene sidechain at C23. The compound of formula (I) showsantiproliferative activity and is most unlikely a substrate forP-glycoprotein and/or Multidrug Resistant Protein.

The compound of formula (I) is a new bafilomycin-like derivativeisolated from liquid fermentation cultures of novel actinomycetes of thegenus Micromonospora.

The compound of formula (I) was extensively characterized for itsability to inhibit tumour cell growth. The dose-dependent inhibition ofthe compound of formula (I) in four selected tumour cell proliferationassays was typical for classical cytotoxics, with very steep and narrowdose-response curves. Initial observation on inhibition of tumour cellproliferation was extended to a wide variety of human tumour cell lines,from ovary, prostate, pancreas, colon and lung origins, with observedIC₅₀ values in the nM range of concentrations.

Multi-drug resistance is considered as one of the major elements in thefailure of anticancer therapeutics in clinical trials. Tumours becomeresistant to therapeutic agents by overexpression of both P-glycoproteinand Multidrug Resistant Protein which represent the major elements ofthe Multidrug Resistant system. Moreover, the constitutive expression ofP-glycoprotein in the intestinal epithelium will, in case of oraladministration, markedly reduce absorption when the compound is asubstrate for the P-glycoprotein. The activity of the compound offormula (I) on P-glycoprotein or Multidrug Resistant Protein-expressingtumour cell lines and respective parental cells, was compared to that ofa known substrate for P-glycoprotein/Multidrug Resitant Protein,adriamycin. The compound of formula (I) inhibited cell proliferation inthe nM range of concentrations regardless of the P-glycoportein and/orMultidrug Resistant Protein expression status. These results stronglysuggest that there is no substantial recognition of the compound offormula (I) by the P-glycoprotein and Multidrug Resistant Protein pumpproteins.

The present novel cancer therapeutic agent shows, like most members ofthis class of compounds, some acute toxicity at higher concentrations.Persons skilled in the art will however be able to determine anappropriate therapeutic range depending upon the condition of thepatient.

Pharmaceutically acceptable addition salts encompass pharmaceuticallyacceptable acid addition salts and pharmaceutically acceptable baseaddition salts. The compound of formula (I) having basic properties maybe converted in its pharmaceutically acceptable acid addition salts bytreating it with an appropriate acid. Appropriate acids comprise, forexample, inorganic acids such as hydrohalic acids, e.g. hydrochloric orhydrobromic acid; sulfuric; nitric; phosphoric and the like acids; ororganic acids such as, for example, acetic, propanoic, hydroxyacetic,lactic, pyruvic, oxalic, malonic, succinic (i.e. butanedioic acid),maleic, fumaric, malic, tartaric, citric, methanesulfonic,ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-amino-salicylic, pamoic and the like acids.

The compound of formula (I) having acidic properties may be converted inits pharmaceutically acceptable base addition salt by treating it with asuitable organic or inorganic base. Appropriate base salt formscomprise, for example, the ammonium salts, the alkali and earth alkalinemetal salts, e.g. the lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e.g. the benzathine,N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like.

The term “acid or base addition salts” also comprises the hydrates andthe solvent addition forms which the compound of formula (I) is able toform. Examples of such forms are e.g. hydrates, alcoholates and thelike.

The structure encompasses several carbonyl groups. These carbonyl groupsmay give rise to what is generally referred to as keto-enol tautomerism.As an example there is shown hereunder the keto-enol tautomers that arein equilibrium. For simple aliphatic ketones there is very little of theenol form present at equilibrium. But in other, instances the enol formcan be present in higher concentration.

Tautomerism is a type of isomerism where the isomers only differ by theplacement of a proton and the corresponding location of the double bond.The isomers which in solution are in equilibrium are called tautomers.

It should be clear that any tautomer of the present structure is meantto be included in the scope of the present application.

This invention also provides a method for inhibiting tumour growth, amethod for the treatment of cancer, by administering an effective amountof the compound of the present invention, to a subject, e.g. a mammal(and more particularly a human) in need of such treatment. Examples ofcancer types which may be inhibited, but are not limited to solid andhaematological cancers, leukemia, skin cancers such as melanoma andbasal cell carcinoma, renal cancer, lung cancer such as small cell lungcancer, non-small-cell lung cancer and alveolar cell carcinoma, coloncancer, breast cancer, prostate cancer, pancreatic cancer, stomachcancer, medullary thyroid cancer, brain cancers and head/neck cancers.

These compounds may also be useful in the treatment ofneurofibromatosis, reumathoid arthritis, restenosis and otherproliferative diseases.

The present compound, in view of its antibacterial characteristics, isalso useful as an antibacterial medicine. This invention also provides amethod for treating bacterial infections, by administering an effectiveamount of the compound of the present invention, to a subject, e.g. amammal (and more particularly a human) in need of such treatment.

The present invention discloses the compound of formula (I) for use as amedicine as well as the use of these compounds of formula (I) for themanufacture of a medicament for inhibiting tumour growth.

In view of its useful pharmacological properties, the subject compoundmay be formulated into various pharmaceutical forms for administrationpurposes.

To prepare the pharmaceutical compositions of this invention, aneffective amount of a particular compound, in base or acid addition saltform, as the active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirably inunitary dosage form suitable, preferably, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules and tablets.

Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. For parenteralcompositions, the carrier will usually comprise sterile water, at leastin large part, though other ingredients, to aid solubility for example,may be included. Injectable solutions, for example, may be prepared inwhich the carrier comprises saline solution, glucose solution or amixture of saline and glucose solution. Injectable suspensions may alsobe prepared in which case appropriate liquid carriers, suspending agentsand the like may be employed. In the compositions suitable forpercutaneous administration, the carrier optionally comprises apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives of any nature in minor proportions,which additives do not cause a significant deleterious effect to theskin. Said additives may facilitate the administration to the skinand/or may be helpful for preparing the desired compositions. Thesecompositions may be administered in various ways, e.g., as a transdermalpatch, as a spot-on or as an ointment.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used in thespecification and claims herein refers to physically discrete unitssuitable as unitary dosages, each unit containing a predeterminedquantity of active ingredient, calculated to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier. Examples of such dosage unit forms are tablets (includingscored or coated tablets), capsules, pills, powder packets, wafers,injectable solutions or suspensions, teaspoonfuls, tablespoonfuls andthe like, and segregated multiples thereof.

Those skilled in the art could easily determine the effective amountfrom the test results presented hereinafter. In general it iscontemplated that a therapeutically effective amount would be from 0.001mg/kg to 100 mg/kg body weight, and in particular from 0.5 mg/kg to 100mg/kg body weight. It may be appropriate to administer the required doseas two, three, four or more sub-doses at appropriate intervalsthroughout the day. Said sub-doses may be formulated as unit dosageforms, for example, containing 0.5 to 500 mg, and in particular 10 mg to500 mg of active ingredient per unit dosage form.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Dose-dependent inhibition of tumour cell proliferation(bioassay) by the compound of formula (I). Human MCF-7 tumour cells weretreated with indicated concentrations of the compound of formula (I) for4 days. Results, expressed as % of control untreated cells, are themean±SE of at least 3 different experiments.

FIG. 1B: Dose-dependent inhibition of tumour cell proliferation(bioassay) by the compound of formula (I). Human HT-29 tumour cells weretreated with indicated concentrations of the compound of formula (I) for4 days. Results, expressed as % of control untreated cells, are themean±SE of at least 3 different experiments.

FIG. 1C: Dose-dependent inhibition of tumour cell proliferation(bioassay) by the compound of formula (I). Human K562/C1,000 tumourcells were treated with indicated concentrations of the compound offormula (I) for 4 days. Results, expressed as % of control untreatedcells, are the mean±SE of at least 3 different experiments.

FIG. 1D: Dose-dependent inhibition of tumour cell proliferation(bioassay) by the compound of formula (I). Human Malme-3M tumour cellswere treated with indicated concentrations of the compound of formula(I) for 4 days. Results, expressed as % of control untreated cells, arethe mean±SE of at least 3 different experiments.

EXPERIMENTAL PART

In the course of a screening program designed to identify new antitumourcompounds (cytotoxics), a new bafilomycin-like metabolite was isolated.The same metabolite was isolated from liquid fermentation cultures oftwo different actinomycete of the genus Micromonospora. Micromonosporaspp. JS1035 was isolated from river sediments from Sombo, Cameroon whileMicromonospora spp. JS1044 was isolated from river sediments fromMundemba, Cameroon. As soon as it was detected that both strainsproduced the same active compound it was decided to further characterizethe compound present in liquid fermentation cultures from Micromonosporaspp. JS1035. A culture of each micro-organism utilised for theproduction of these new macrolide have been deposited in the Laboratoryfor Microbiology in the Belgian Coordinated Collections of Microorganismat the University of Gent under accession number LMG P-21525 forMicromonospora spp. JS1035 and under accession number LMG P-21526 forMicromonospora spp. JS1044. These deposits have been made under theprovisions of the Budapest Treaty and all restrictions on theavailability thereof to the public will be irrevocably maintained upongranting of a patent on this application.

General

Fermentation and Isolation

Liquid fermentation cultures of Micromonospora spp. JS1035 wereextracted with ethyl acetate to yield the crude extract that wassubsequently purified using counter current partition chromatography(CPC). Pure material for structure elucidation was obtained after twoconsecutive CPC fractionations using different conditions. Mass spectralanalysis (see Table 1) of this material indicated a molecular weight of853 and a molecular formula of C₄₇H₆₇NO₁₃. TABLE 1 Physico-chemicalproperties of the compound of formula (I). Appearance Light brown solidMelting Point 114-120° C. [α]²⁵ _(D) −38.0 (c 0.1, MeOH) MolecularFormula C₄₇H₆₇NO₁₃ Pos. ESI*-MS 854 (M + H)⁺ HRFAB*-MS (m/z) Found:876.4514 Calcd: 876.4510 (for C₄₇H₆₇NO₁₃Na) UV (MeOH) λ_(max) nm (ε)233, 286, 345 IR* ν_(max)(film) cm⁻¹ 3408, 2923, 1717, 1687, 1614, 1247,1097, 751*ESI: Electron Spray Ionisation, HRFAB: High Resolution Fast AtomBombardment, IR: Inversion RecoveryStructure Determination

The NMR data for the compound of formula (I) was initially obtained inCDCl₃. However, it was soon discovered that the compound of formula (I)was not stable in this solvent. Subsequent NMR work was performed usingacetone-d₆ and yielded a complex proton NMR spectrum. A full set of NMRexperiments including ¹³C NMR, Distortionless Enhancement byPolarization Transfer (DEPT), Correlated Spectroscopy (COSY),Heteronuclear Multiple Quantum Coherence (HMQC), and HeteronuclearMultiple Band Coherence (HMBC) was obtained.

Assembling the partial structures for the compound of formula (I) wasstraightforward. The NMR data (Table 2) for the macrolide, C16-C23, andC1′-C4′ were in good agreement with published data. (Werner G. et al. J.Antibiotics 37: 110-117, 1984) Attachment of the new hexadiene sidechain(C24-C29) was accomplished by careful study of the correlations betweenrelevant atoms. For instance, H24 showed an HMBC correlation to C22 andC23, and H25 also showed an HMBC correlation to C23. The COSY data werealso consistent with placement of the new sidechain at C23. Several ¹³CNMR signals (C5′, C6′, and C9′) corresponding to the cyclopentenone ringwere obscured in the ¹³C NMR spectrum, probably due to the presence ofresonance structures between the vinylic OH and the carbonyl group.

Other published reports detail the stereochemistry for otherbafilomycins and other members of this class (O'Shea M. et al J.Antibiotics 50: 1073-1077, 1997). Molecules in this class have the samestereochemistry for the lactone ring, the tetrahydropyrin ring, and theC16-C18 linkage. All appear to possess a hydrogen-bonding network thatincludes C19OH, C17OH, and C1=O. This network effectively defines theconfiguration of the side chain. Based on the analysis of couplingconstant, chemical shift, and Rotating Frame Overhauser EffectSpectroscopy (ROESY) data, the proposed stereochemistry of the compoundof formula (I) is consistent with this conformation. The new hexadienesidechain at C23 was assigned as alpha to the ring based on two keyROESY correlations. There is a correlation between H23 and CH₃-36 andalso between H23 and H21. These correlations would be unlikely unlessH23 was positioned above the ring. TABLE 2 ¹H and ¹³C NMR chemicalshifts for the compound of formula (I)^(a) δ_(C) (ppm) M δ_(H) (ppm) 1167.57 s 2 141.97 s 3 133.35 d 6.63(1H, s) 4 132.74 s 5 145.55 d5.94(1H, d, 8.7) 6 37.85 d 2.52(1H, m) 7 80.17 d 3.28(1H, br d, J 5.4) 841.93 d 1.86(1H, m) 9 42.27 t 2.12(1H, m), 2.03(1H, m) 10 144.98 s 11125.19 d 5.80(1H, d, 10.7) 12 134.53 d 6.67(1H, dd, 15.0, 10.8) 13126.95 d 5.12(1H, m) 14 82.98 d 4.07(1H, m) 15 77.01 d 4.96(1H, dd, 8.4,1.3) 16 38.23 d 2.13(1H, m) 17 70.80 d 4.11(1H, m) 18 42.27 d 1.84(1H,m) 19 100.26 s 20 40.22 t 2.35(1H, dd, 11.7, 4.8), 1.34(1H, m) 21 75.11d 5.08(1H, m) 22 41.81 d 1.50(1H, m) 23 75.34 d 4.14(1H, m) 24 130.81 d5.50(1H, dd, 15.1, 7.6) 25 133.51 d 6.18(1H, dd, 15.5, 15.4) 26 129.72 d5.99(1H, dd, 15.1, 10.5) 27 137.21 d 5.70(1H, m) 28 26.16 t 2.09(2H, m)29 13.73 q 0.97(3H, d, 2.8) 30 14.13 q 1.94(3H, br s) 31 17.62 q1.03(3H, d, 7.0) 32 22.20 q 0.90(3H, d, 6.8) 33 20.33 q 1.93(3H, br s)34 10.02 q 0.85(3H, m) 35 7.2 q 1.00(3H, d, 2.5) 36 11.61 q 0.85(3H, m)2-OCH3 59.94 q 3.50(3H, s) 14-OCH3 55.63 q 3.23(3H, s) 1′ 168.14 s 2′134.97 d 7.57(1H, d, 15.3) 3′ 132.97 d 6.82(1H, d, 15.3) 4′ 165.00 s 5′^(b) s 6′ ^(b) S 7′ 27.75 t 2.52(2H, m) 8′ 37.31 t 2.52(2H, m) 9′ ^(b) s^(a)δ in ppm, J value in Hz. Data were recorded at 500 and 125 MHz,respectively in acetone-d6.^(b)Signal not observed.Biological Activity

A panel of cell proliferation assays (referred to herein as “bioassays”)based on the inibition of cell proliferation in selected human tumourcell lines (mammary MCF-7, colon HT-29, leukemia K562/C1,000 andmelanoma Malme-3M) was used to identify antiproliferative activities inthe different samples throughout the purification procedures. Extractsfrom strain JS1035 grown in four different media were tested inbioassays; the one that contained most of the antiproliferative activitywas selected for further study. A 1:1,000-dilution of the active extractresulted in an almost complete inhibition of cell proliferation in thethree cell lines tested (melanoma Malme-3M cell line was omitted at thisstage). From this initial crude extract, collected fractions fromcounter current partition chromatography were individually analyzed onMCF-7 cell proliferation. A major single fraction was shown to containantiproliferative activity, which was then further purified by HPLC. Asingle HPLC peak was found to contain antiproliferative activities asmeasured by inhibition of cell proliferation in the bioassay. This peakwas used to initiate structure elucidation of the active compound offormula (I). Subsequent structure work was completed using materialisolated with two consecutive CPC steps.

The purified compound of formula (I) was extensively characterized forits ability to inhibit tumour cell growth. As indicated in FIG. 1A-D,the dose-dependent inhibition of the compound of formula (I) in the fourselected tumour cell proliferation assays was typical for classicalcytotoxics, with very steep and narrow dose-response curves. Thecompound of formula (I) exhibited similar potency for inhibition oftumour cell proliferation in the nM range of concentrations, with aslightly higher activity on colon HT-29 carcinoma cells (sub-nanomolarconcentrations) (Table 3). Initial observation on inhibition of tumourcell proliferation was extended to a wide variety of human tumour celllines, from ovary, prostate, pancreas, colon and lung origins, withobserved IC₅₀ values in the nM range of concentrations (Table 4).

The activity of the compound of formula (I) on P-glycoprotein- orMultidrug Resistant Protein-expressing tumour cell lines and respectiveparental cells, K562/C1,000+ and K562A7, COR-L23/R and COR-L23, wascompared to that of a known substrate for P-glycoprotein/MultidrugResistant Protein, adriamycin. As indicated in Table 5, the compound offormula (I) inhibited cell proliferation in the nM range ofconcentrations regardless of the P-glycoprotein and/or MultidrugResistant Protein expression status (no significant difference betweenIC₅₀ values). On the contrary, K562/C1,000+ and CORL23/R cells exhibiteda 250-fold and 20-fold resistance to cell killing by adriamycin,respectively, as compared to their respective parental cell lines. Theseresults strongly suggested that there is no substantial recognition ofthe compound of formula (I) by the P-glycoprotein and MultidrugResistant Protein pump proteins. TABLE 3 Inhibition of tumour cellproliferation by the compound of formula (I). Sensitivity to cellkilling by the compound of formula (I) was assessed in breast MCF-7,colon HT-29, leukemia K562/C1,000 and melanoma Malme-3M cells. Cellviability was determined after a 4-day incubation in the absence(control) or presence of increasing concentrations of the compound offormula (I). Results, means ± SE from at least three separateexperiments, are expressed as IC₅₀ values (in nM). Compound Cell Linesof formula (I) MCF-7 1.92 ± 0.35 HT-29 0.61 ± 0.04 K562/C1,000 9.89 ±7.35 Malme-3M 7.96 ± 2.73

TABLE 4 Effect of the compound of formula (I) on human tumour cellproliferation. Activity of the compound of formula (I) on tumour cellproliferation was assessed on several human tumour cell lines fromvarious origins. As indicated in the Materials and Methods section, a4-day incubation period in the absence (control) or presence ofincreasing concentrations of the compound of formula (I) was followed bycell viability determination using MTT staining method. Results, means ±SE from two separate experiments, are expressed as IC₅₀ values (in nM)which represent the concentration of compound required to inhibit cellproliferation by 50% . Compound Cell Line Tissue origin of formula (I)A2780 ovary 1.37 ± 0.13 DU 145 prostate 1.95 ± 0.30 Capan-1 pancreas6.93 ± 3.39 HCT 116 colon 1.56 ± 0.03 LoVo colon 5.98 ± 5.52 H1299 lung1.46 ± 0.13

TABLE 5 Is the compound of formula (I) a substrate forP-glycoprotein/Multidrug Resistant Protein? Sensitivity to cell killingby the compound of formula (I) and adriamycin was assessed in humanK562A7 parental leukemia cells and P-glycoprotein overexpressingcounterpart, K562/C1,000+ cells; as well as parental small cellcarcinoma COR-L23 cells, and Multidrug Resistant Protein overexpressingcounterpart, COR-L23/R cells. Cell viability was determined after a4-day incubation in the absence (control) or presence of increasingconcentrations of the compound of formula (I). Results, mean ± SE fromat least two separate experiments, are expressed as IC₅₀ values (in nM).Compound Cell Line of formula (I) Adriamycin K562A7 14.08 ± 1.25  26.78± 5.82 K562/C1,000+ 29.34 ± 1.80 7,829 ± 806 COR-L23  5.13 ± 0.91  36.57 ± 19.09 COR-L23/R  6.90 ± 0.24   676 ± 261

EXAMPLE 1

Structure Determination

¹H and ¹³C NMR spectra were recorded at 300K on a Bruker Avance DRX 500spectrometer operating at 500 and 125 MHz, respectively. Standard pulsesequences were used for DEPT, COSY, HMQC, and HMBC experiments. ROESYdata was acquired using a spinlock pulse of 300,000 μs (at 22 dB).Melting points were determined using an Electrothermal 9100 meltingpoint apparatus and are uncorrected. Inversion Recovery (IR) data wasacquired using a Perkin Elmer 1600 Series FYIR spectrophotometer. APerkin-Elmer 243B polarimeter was used to procure optical rotation data.High resolution MS data was obtained using a Micromass 70SEQ TandemHybrid Mass Spectrometer. CPC was performed on a P.C., Inc. high speedcountercurrent chromatograph, equipped with an Ito multilayer coilcolumn interfaced with a Waters 991 Photodiode Array detector(monitoring at 270 nm).

Fermentation and Isolation

The actinomycete strain JS1035 was isolated from river bottom sedimentcollected near Sombo, Cameroon. The seed medium contained glucose 20 g,Pharmamedia (Trader's Protein) 15.0 g, yeast extract 5.0 g, CaCO₃ 4.0 g,(NH₄)₂(SO₄) 3.0 g, ZnSO₄.7H₂O 0.003 g in 1 L distilled water, pHadjusted to 6.5. The production medium contained glucose 20.0 g, dextrin50.0 g, Pharmamedia (Trader's Protein) 30.0, yeast extract 1.0 g, CaCO₃5.0 g, CoCl₂.6H₂O 0.001 g in 1 L distilled water, pH adjusted to 7.0.The media were dispensed in 25 or 30 mL aliquots to 250 mL flasks andautoclaved for 15 min. Frozen spore stocks (1.0 mL) were used toinoculate starter cultures (25 mL seed media per 250 mL Erlenmeyerflask). Starter cultures were incubated at 28° C. and ˜75% humidity onan orbital shaker (2″ throw, 250 rpm) for 48 h. The starter cultures (1mL) were used to inoculate 200 production media flasks (30 mL productionmedia per 250 mL Erlenmeyer flask), which were then incubated for 6 daysat the above conditions.

Ethyl acetate (22.5 mL) was added to each flask, and the flask contentswere pooled into 500 mL centrifuge bottles. Each bottle was shakenvigorously and centrifuged at 6000×g for 8 minutes. The ethyl acetatewas removed from each bottle and dried over anhydr. Na₂SO₄. Ethylacetate was again added to the centrifuge bottles to a total volume ofapproximately 450 mL, and the process was repeated. The extract wasdried under reduced pressure to yield a crude extract (5.4 g).

An aliquot (400 mg) of the crude extract was subjected to CPC. Thesolvent system consisted of an equilibrated mixture of n-hexane, EtOAc,MeOH, and water (1:3:3:3, v/v/v/v). The lower phase was used as thestationary phase, and the upper phase served as the mobile phase whichwas pumped at 3 mL/minute. The column rotation speed was 800 rpm. Thecrude extract was prepared by dissolving it in a mixture of upper andlower phases. The material was then loaded onto the column. The firstpeak to elute was collected and dried to yield 303 mg of purifiedmaterial. This purified material was again subjected to CPCchromatography using modified conditions. The modified solvent systemconsisted of an equilibrated mixture of water, MeOH, and n-hexane(1:9:10, v/v/v). Again, the lower phase was used as the stationaryphase, and the upper phase served as the mobile phase. After 90 minutes,the upper and lower phases were switched and the first peak to eluteafter the solvent change was collected and dried to yield 9.2 mg of thepure compound of formula (I).

Initially, the compound of formula (I) was isolated using HPLC followingthe first CPC separation. In that case, the active CPC fraction wasfurther separated using reversed-phase HPLC (LiChrospher,10 μm, 10×25mm, C₁₈). A linear gradient starting at 95:5 CH₃CN:H₂O to 100% CH₃CNover 25 minutes was used to isolate the compound of formula (I). A 5-mgHPLC injection yielded 0.5 mg of compound (I).

Drugs

All samples tested were dissolved in DMSO and further dilutions(typically 1:1,000) were made in culture medium, with final DMSOconcentrations never exceeding 0.1% (v/v) in cell proliferation assays.MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuminner salt] and PMS [phenazine methosulfate] were dissolved inphosphate-buffered saline (PBS).

MTT and MTS are both tetrazolium salts widely used in cell proliferationand toxicity tests. Upon entry in a viable cell, these tetrazolium saltsare (bio)reduced to an intensely colored formazan derivative. In case ofMTT, the formed formazan is crystalline and needs to be dissolved beforecolorimetric quantification. MTS needs an intermediate electron acceptor(PMS) for its efficient reduction into a soluble formazan end productand is used for suspension cell lines.

Cell Lines and Cell Culture

The human MCF-7 mammary adenocarcinoma cells were cultured in Dulbecco'sModified Eagle Medium (DMEM) supplemented with 2 mM L-glutamine, 1 mMNaPyruvate, 50 μg/ml gentamicin and 5% fetal calf serum. The human HT-29colon carcinoma cells were cultured in Mc Coy's 5a medium supplementedwith 2 mM L-glutamine, 50 μg/ml gentamicin and 5% fetal calf serum. Thehuman Malme-3M melanoma cells were cultured in DMEM medium supplementedwith 2 mM L-glutamine, 1 mM NaPyruvate, 50 μg/ml gentamicin and 10%fetal calf serum. The human DU 145 prostate carcinoma cells werecultured in DMEM medium supplemented with 2 mM L-glutamine, 1 mMNaPyruvate, 50 μg/ml gentamicin and 10% fetal calf serum. The humanCapan-1 pancreatic adenocarcinoma cells were cultured in DMEM mediumsupplemented with 2 mM L-glutamine, 1 mM NaPyruvate, 50 μg/ml gentamicinand 10% heat inactivated fetal calf serum. The human HCT 116 colorectalcarcinoma cells were cultured in Mc Coy's 5a medium supplemented with 2mM L-glutamine, 50 μg/ml gentamicin and 10% fetal calf serum. The humanH1299 non-small lung carcinoma cells were cultured in RPMI 1640 mediumsupplemented with 2 mM L-glutamine, 1 mM NaPyruvate, 10 mM HEPES and 10%heat inactivated fetal calf serum. All these cell lines were obtainedfrom ATCC [American Type Culture Collection, Manassas, Va., USA]. Thehuman A2780 ovarian carcinoma cell line was a kind gift from Dr. T. C.Hamilton [Fox Chase Cancer Centre, Pennsylvania, USA] and was culturedin RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 μg/mlgentamicin and 10% fetal calf serum. The human LoVo colon adenocarcinomacell line was a kind gift from Dr. M. Grandi [Pharmacia-Fice, Nerviano,Milano, Italy] and was cultured in HAM's F12 medium supplemented with 2mM L-glutamine, 50 μg/ml gentamicin, 1% Vitamins (BME) and 10% fetalcalf serum. Cells were routinely kept as monolayer cultures at 37° C. ina humidified 5% CO₂ atmosphere. The human K562/C1,000 leukemia cell linewas a kind gift by Dr. H. Heyligen [Dr. Willems Instituut, Diepenbeek,Belgium] and is a P-glycoprotein expressing cell line obtained byculturing the cells at increasing concentrations of colchicine. Thecells were kept as a suspension culture at 37° C. in a humidified 5% CO₂atmosphere in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50μg/ml gentamicin and 5% fetal calf serum. The human K562A7 leukemia cellline was a kind gift by Dr. H. Heyligen and is the parent, drugsensitive cell line of the K562/C1,000 cells. The K562/C1,000+ cell linewas obtained by culturing the K562/C1,000 cells for an additional 2weeks with 1 μM colchicine. This treatment induces higher levels ofP-glycoprotein and mRNA and a more stable resistance profile. The cellswere kept as a suspension culture at 37° C. in a humidified 5% CO₂atmosphere in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50μg/ml gentamicin and 5% fetal calf serum. The human large cell lungcancer cell lines were obtained from ECACC (European Collection of CellCultures, Salisbury, U.K.). COR-L23 is the parent, drug sensitive cellline and COR-L23/R is the Multidrug Resistant Protein-expressingMultidrug Resistant-variant, developed by continuous step-wise in vitroincubation of COR-L23 cells with increasing concentrations ofdoxorubicin. The culture medium for both cell lines consisted of RPMI1640 supplemented with 2 mM L-glutamine, 50 μg/ml gentamicin and 10%fetal calf serum. Cells were routinely kept as monolayer cultures at 37°C. in a humidified 5% CO₂ atmosphere.

All media and supplements were obtained from Life Technologies,Merelbeke, Belgium. Cells were free of mycoplasma contamination asdetermined using the Gen-Probe Mycoplasma Tissue Culture kit[BioMérieux, Brussel, Belgium].

Cell Proliferation Assays

Cell proliferation assays were performed as described (A criticalassessment of the use of microculture Tetrazolium assays to measure cellgrowth and function (Mossman, T. J. Inmunol Meth 65: 55-63, (1983)).

In case of adherent cell lines, cells were seeded in Falcon/NUNC 96-wellculture plates [Life Technologies, Merelbeke, Belgium] and allowed toadhere to the plastic for 18-48 hr. Medium was then changed and drugsand/or solvents were added. Following 4-day incubation, cell density wasassessed using a MTT-based assay measuring absorbance at 540 nm using aEmax 96-well spectrophotometer [Sopachem, Brussel, Belgium]. For theexperiments with the suspension culture, K562A7, K562/C1,000 andK562/C1,000+ cells were seeded in Falcon® 96-well culture plates. Testdrugs and/or medium were added immediately after seeding. Following4-day incubation, cell growth was assessed using a MTS/PMS based assaymeasuring absorbance at 490 nm using an Emax 96-well spectrophotometer.

The antiproliferative activity is calculated as percentage of remainingviable cells in treated versus control (untreated cells) conditions.Results are expressed as IC₅₀ values which represent the concentrationof compound required to inhibit cell proliferation by 50%. Within anexperiment, the result for each experimental condition is the mean of atleast 3 replicate wells.

1. A compound of formula (I)

a tautomer thereof or a pharmaceutically acceptable salt thereof
 2. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier, and as active ingredient a therapeutically effective amount ofa compound as described in claim
 1. 3. A process for preparing apharmaceutical composition wherein a therapeutically effective amount ofa compound as claimed in claim 1 is intimately mixed with apharmaceutically acceptable carrier.
 4. A process for the production ofa compound of formula (I) as defined in claim 1, comprising cultivatinga strain of a microorganism capable of producing a compound of formula(I), recovering the compound of formula (I) from the cultured broth, andoptionally, salifying the recovered compound.
 5. A process according toclaim 4, wherein the microorganism is a Micromonospora species.
 6. Acompound according to claim 1 for use as a medicine.
 7. The use of acompound according to claim 1 in the manufacture of a medicament forinhibiting cancer or bacterial growth.
 8. A method for the treatment ofcancer, comprising administering to a mammal in need of such treatmentan effective amount of a compound of formula (I) as defined in claim 1.9. A compound isolated from Micromonospora spp JS1035 deposit No LMGP-12525 having antitumour activity.
 10. A compound as defined in claim 9wherein the compound has a molecular weight of 853 and a molecularformula of C₄₇H₆₇NO₁₃.
 11. A microorganism wherein said organism isdeposited under deposit No. LMG P-12525.
 12. A microorganism whereinsaid organism is deposited under deposit No. LMG P-12526.